Polyredox couples in analyte determinations

ABSTRACT

Enzyme or substrate determinations can be achieved by employing an organic second substrate, which produces a product which may be coupled with a metal inorganic redox couple capable of interacting with and affecting the potential of a metal electrode on a semiconductor surface. Particularly, hydrogen peroxide or indoxyl phosphate are coupled with iron- or ruthenium-containing ionic redox couples for determination with a noble metal electrode.

This is a continuation of application Ser. No. 07/952,465 filed Sep. 28,1992, now abandoned which is a continuation of application Ser. No.07/497,988, filed Mar. 23, 1990 now abandoned.

TECHNICAL FIELD

The field of this invention is the use of bioensors to detect redoxreactions involving enzymes.

BACKGROUND

For the most part, assays involving the detection of enzyme activity,either for measuring enzyme substrate, enzyme, or the enzyme as a labelhave depended upon colorometric or fluorimetric detection. However,there has been a continuous and expanding interest in electricaldetection using either amperometric or potentiometric methods. Thesemethods require a redox couple which can be detected at an electrode.

There are many constraints in developing a diagnostic system.Compositions must be developed which allow for the transport ofelectrons from the enzyme or a product of the enzyme. The variousmaterials which are employed must not interfere with each other, so thatneither the enzyme reaction, nor the transfer of electrons to theelectrode, are adversely affected. Because in many instances the analyteis present in very low concentration, it is important that theindividual reaction related to the amount of analyte be very rapid,desirably diffusion controlled. In this circumstance, there will be arapid response for each enzymatic event.

It is therefore of interest to devise compositions which may be used inthe electrical detection of enzymatic reactions, by providing for rapidand efficient transfer of electrons to an electrical sensor as a resultof enzymatic reaction.

RELEVANT LITERATURE

Frew and Mill, Anal. Chem. (1987) 59:933A 944A, provide a review ofelectrochemical biosensors. Josephy et al., J. Biol. Chem. (1982)257:3669-3675 describe the use of tetramethylbenzidine as a horse radishperoxidase substrate. References of interest employing biosensors inenzyme immunoassays include Heineman et al., Anal. Proc. (1987)24:324-325; Alwis and Wilson, Anal. Chem. (1987) 59:2786-2789; Gyss andBoutdillon, Anal. Chem. (1987) 59:2350-2355; Sayo et al., J. of Chromat.(1987) 417:129-134; Lunte et al., "Immunoassay by HPLC and FlowInjection Analysis with Electrochemical Detection" In Proceedings of theSymposium on Electrochemical Sensors for Biomedical Applications, ed.Li, Vol. 86-14, Electrochemical Soc., Inc., N.J., 1986, pp. 129-138;Wehmeyer et al., Anal. Chem. (1986) 58:135-139; Janata, Anal. Proc.(1987) 24:326-328; Weetall and Hotaling, Biosensors (1987) 3:57-63;Davis, Biosensors (1986) 2:101-124; and EPA 85/114836.3.

SUMMARY OF THE INVENTION

Compositions are provided for determining enzymatic activity in a mediumemploying an organic substrate coupled with an inorganic redox couplefor efficient transfer to a metal electrode.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for detecting enzymatic reactionsemploying a metal electrode. The methods employ a combination ofreagents, where the first reagent provides for a rapid efficientreaction with the enzyme to produce a product capable of reacting withthe second reagent, where the second reagent may be determinedamperometrically or potentiometrically. The method may involve directdetection of the enzyme, use of an enzyme as a label for detection ofligands, or use of an enzyme to detect enzyme substrate.

The enzymes to be detected are hydrolases, particularly phosphatases, oroxidoreductases, particularly peroxidases, which may be used bythemselves or in conjunction with oxidases. The oxidases can produce aproduct, particularly hydrogen peroxide, with which the peroxidase mayreact to produce another product which reacts with the second reagent.Oxidases which may be coupled with the peroxidase include glucoseoxidase, xanthine oxidase, uricase, cholesterol oxidase, and the like.

The hydrolases, particularly esterases or saccharidases, such asβ-galactosidase, can provide hydrolysis of indoxyl esters,-where theacid group may be organic or inorganic, including carboxylates,phosphates, sulfates, etc., or indoxyl glycosidyl ethers, such asβ-galactosidyl, β-glucosidyl, etc.

The second reagent is an inorganic redox couple, normally employing aniron or ruthenium couple. The iron may be in any convenient form,particularly coordinated, such as with hexacyanoferrate, e.g. ferroorferricyanide, ferrocene, or other stable form of iron, capable ofundergoing one electron transfer. The ruthenium will also be coordinatedwith a variety of ligands, particularly nitrogen ligands, such aspyridine, substituted pyridines, bis-pyridines, pyridine, amino,substituted amino, or the like. The ruthenium preferably will beemployed in the divalent state, coordinated with 5 NH₃ ligands and 1pyridine ligand.

Various metal electrodes may be employed, particularly inert or noblemetal electrodes, such as gold, rhodium, platinum, etc. As will bediscussed, the metal electrode may be used in relation to thesemiconductor electrode as is described in application Ser. No. 072,168,filed Jul. 10, 1987, which disclosure is incorporated herein byreference.

The first reagent will be selected so as to be able to react rapidly andefficiently with the enzyme to produce a product which will have anelectromotive potential so as to be capable of reacting with the secondreagent. Thus, the first and second reagents are related in that theelectromotive potential of the product of the enzymatic reaction must beat a higher potential than the electromotive potential for the inorganicredox couple.

For use with peroxidases, benzidine or benzidine derivatives can beemployed as the first reagent with advantage, where the benzidine willusually be not more than about 30 carbon atoms, usually not more thanabout 20 carbon atoms, and may have from 0 to 4, usually from 0 to 2other heteroatoms as substituents, where the heteroatoms will usually beoxygen or nitrogen, normally present as oxy, oxo or amino, where theoxygen and nitrogen may be substituted with hydrogen or alkyl of from 1to 3, usually from 1 to 2 carbon atoms. Generally, there will not bemore than 4 substituents bonded to annular carbon atoms, usually notmore than 2 substituents bonded to annular carbon atoms, other than theamino groups of the benzidine. Illustrative substituents include methyl,ethyl, hydroxyl, methoxy, methylamino, etc.

Indoxyl esters will have the ester group at the 3 position and may beotherwise substituted or unsubstituted, usually unsubstituted, wheresubstituents may be alkyl of from 1 to 3 carbon atoms, oxy of from 0 to3 carbon atoms, amino of from 0 to 3 carbon atoms, or halogen, where theethers and substituted amino groups will be substituted by alkyl of from1 to 3 carbon atoms, usually of from 1 to 2 carbon atoms. Halogensubstituents may be chlorine, fluorine, or bromine groups, usually 1 to3, and preferably 2 groups, per indoxyl molecule. As indicatedpreviously, a variety of esters may be employed, which includephosphate, acetate, lactate, sulfate, or the like, or glycosidyl ethers.

Depending upon the purpose of the reaction, various protocols will beemployed, as well as various concentrations. For directly measuring anenzyme, the enzyme concentration may be varied widely, depending uponthe sample. Usually, the enzyme will be present in from about 0 to 1 μg,more usually from about 0 to 10 ng. In addition, this will varydepending upon whether a peroxidase or hydrolase is involved, with theperoxidase being from about 0.1 pg to 10 ng, while the hydrolase willgenerally be from about 1 pg to 100 ng.

The benzidine compound will usually be present in about 50 μM to 1 mM,more usually in 100 to 400 μM. The amount of benzidine will be selectedso as to be non-rate limiting, so as to provide substantially a V_(max).The indoxyl compound will also be present in non-limiting amountsgenerally being present in 0.1 to 2 mM.

The inorganic redox couple generally will be at a concentration in therange of about 1.0 to 1000 μM, more usually from about 10 to 200 μM.

The solution will be buffered to optimize substantially the enzymaticactivity, generally having a pH in the range of about 4 to 12, moreusually from about 5.5 to 10 depending upon the pH optimum of the enzymewith the substrates employed. Various buffers may be employed, whichinclude tris-(hydroxymethyl) methylamine (Tris), phosphate, borate, orthe like, usually employing a buffer suitable for the particular enzymesystem.

For measuring the enzyme, the various components may be brought togetherin an appropriately buffered medium in contact with the metal electrodeand the rate of change of redox potential in the medium determined.

Where the enzyme is a label, a wide variety of protocols are available.Various assay compositions are commercially available, involvingheterogeneous protocols (a separation step) or homogeneous protocols (noseparation step). Assay kits are sold which are generally referred to asELISA or under the trademark EMIT. In performing an ELISA, the mediumcontaining the enzyme conjugate is separated from the enzyme conjugatewhich has become bound to a support. Modulation of binding of enzymeconjugate is due to a competition between a complementary specificbinding pair member bound to a support, and an analyte in solution.Alternatively, in a sandwich assay, the analyte acts as a bridge betweena complementary specific binding pair member, which is attached to thesupport, and the enzyme conjugate. Thereby, the analyte also modulatesthe binding of enzyme conjugate. Various protocols have appeared in thepatent literature and may be exemplified by such patents as U.S. Pat.No. 4,376,110.

For homogeneous enzyme immunoassays, the various reagents may be broughttogether, where binding of the complementary specific binding pairmember to the enzyme conjugate results in a change in enzymaticactivity. Thus, in this instance, one need not separate the bound fromfree enzyme conjugate.

In the heterogeneous assay, the subject reagents may be added to eitherthe surface bound enzyme or to the separated supernatant fordetermination of enzymatic activity. Enzymatic activity is thenmonitored with a metal electrode in accordance with the subjectinvention.

Illustrative of the subject method is the determination of cholesterol,where the cholesterol may be present in a variety of forms, such as HDL,LDL, VLDL or uncomplexed. The cholesterol serves as a substrate forcholesterol oxidase to form hydrogen peroxide, which acts as a substratefor horseradish peroxidase.

The metal electrode may be any type of metal electrode, wire, wafer,grid, or the like. Of particular interest in the subject invention isthe use of a metal electrode which is in electrostatic proximity contactwith a semiconductor. This device is described in U.S. application Ser.No. 072,196, filed Jul. 10, 1987, whose disclosure is incorporatedherein by reference. The apparatus of U.S. Pat. No. 4,591,550 also maybe used where such device is modified by affixing to the insulatingsurface a thin metal film to serve as the metal electrode, so that aplurality of assays may be carried out, where each of the metalelectrodes bound to the insulating surface are insulated one from theother. Any means of insulation may be used such as, separating thesample solutions associated with each of the metal electrodes withdividers or using gelled samples which are insulated one from the other,or the like.

The metal electrode will be present on an insulating layer, e.g. siliconoxide or silicon oxynitride with silicon, of about 0.1 to 10 mmthickness. The metal electrode will generally have a surface area ofabout 0.1 to 5 mm² and about 0.1 to 1000 μ thickness. The electrode maybe electrodeposited or affixed with an adhesive which is stable underthe conditions to which it is subjected. For example, the electrode maybe 0.5 μ of gold over 300 Angstroms of chromium over 1000 Angstroms ofsilicon nitride over 300 Angstroms of silicon oxide over n-type silicon.Any surface of the semiconductor which is exposed to the medium will beprotected with an insulating coat, e.g. 1000 Angstroms silicon nitrideover 100 Angstroms of silicon oxide.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1

Detection of Horseradish Peroxidase

The apparatus employed is described in European patent application Ser.No. 87.305456.3, filed Jun. 19, 1987. The particular constructionemployed for the following tests is as follows:

The gold layer of about 4.2 mm diameter was placed on a 2.5 mm×5.0 mmrectangular silicon chip which is positioned in a reading chambercontaining a platinum controlling electrode and an Ag/AgCl referenceelectrode. A membrane with the enzyme of interest bound to it orincluded in it is positioned over the gold electrode and a plasticplunger is pressed against the membrane to restrict diffusion ofreagents from the localized environment of the membrane.

The silicon chip is fabricated from a 4-inch diameter wafer of N <100>silicon of approximately 10 to 15 ohm-cm resistivity. The insulator iscomposed of approximately 340 Angstroms of silicon oxide adjacent to thesilicon and overlaid with 1000 Angstroms of silicon nitride deposited bychemical vapor deposition from a reaction of dichlorosilane and ammoniaat about 800° C. in a low pressure chamber. The wafers are subsequentlyannealed in a hydrogen ambient at 1050° C. for 1 hour. The ohmic contactto the silicon chip is made by evaporating approximately 0.5 μ ofgold--1% arsenic onto the (bare) etched back surface of a silicon wafer,etching away the gold from regions where light penetration is desiredand then alloying the gold into the silicon at 450° C. The sensing goldlayer next is deposited over the silicon nitride insulating layer byevaporation in a low pressure chamber of first 300 Angstroms of chromiumfollowed by 5000 Angstroms of gold. The 4.2 mm circles are then formedby protection with a positive photoresist followed by etching first thegold and then the chromium by means of standard metal-etchingtechniques.

The following reagents were employed:

1) horseradish peroxidase;

2) tetramethylbenzidine (TMB) (20 mg/ml in 95% ethanol);

3) peroxidase buffer: 0.1 M NaAcetate, 1 mM EDTA, 7.5% ethanol, pH 5.5;

4) potassium retro- or ferricyanide, each 100 mM in water.

5) Ruthenium Pentaamine Pyridine Perchlorate, 4.4 mM in water.

A redox solution was-prepared comprising TMB 200 μM, Ruthenium ⁺ 2pentaamine pyridine 25 μM, H₂ O₂, 250 μM, in peroxidase buffer.Concentrations of horseradish peroxidase on a nitrocellulose supportwere introduced into the chamber and readings taken by employing a sweep-0.5 to -1.5 volts, gain 16, sweep time 0.4 sec. At 800 pg HRP, theobserved result was -3046 μV/s, at 91 pg, -1784 μV/s, at 6 pg, and -327μV/s at 0 ng. It is evident that by employing the benzidine substrate,TMB, and an inorganic redox couple, ruthenium pentamine pyridine, it ispossible to rapidly detect (within 60 secs) -1 pg of HRP, 2.5×10⁻¹⁷moles of HRP, or 16×10⁶ molecules of HRP.

In the next study, detection of microorganisms was demonstrated. Asolution was prepared of N. meningitidis (4×10⁹ cells/ml). A monoclonalantibody-HRP conjugate was prepared and employed in 1:200 dilution in40% fetal bovine serum (FBS). The membrane filter (polycarbonate) had a0.2 μm pore 0.2 ml of sample containing varying concentrations of cellswas filtered onto the filter followed by incubation with 50 μl ofconjugate, after which time the filter was washed 2× with PBS and 1×with NaCl (0.9%)-Tween-20 (0.05%). To the substrate buffer (0.1 M NaAc,1 mM EDTA, 5% ethanol, pH 5.5, 0.1% Triton X-100) was added: TMB (200μM); ferrocyanide (100 μM); and hydrogen peroxide (250 μM). Followingthe procedure described above, the signal was determined for 8×10³,8×10⁴ and 8×10⁵ cells. The observed signal for 8×10³ cells was 4.6× thenegative signal (-80 μV/s) -369 μV/s, the 8×10⁴ cells' signal was -2231,and the 8×10⁵ cells signal was -10080.

When ferrocene was employed in place of the ferrocyanide at aconcentration of 200 μM and 8×10³ cells, the ratio was 3.5× neg, whilewith 800 cells, the ratio was 1.36× neg.

In the next study, ferri-/ferrocyanide was used as the inorganic redoxpair in conjunction with TMB.

The reaction solution was 10 μM in potassium ferrocyanide, 0.1 MNaAcetate, pH 5.5, 1 mM EDTA, 0.1% Triton X-100, 5% ethanol, 250 μMhydrogen peroxide, 200 μM TMB.. The HRP conjugate (Lot No. 7-6-87A) wasdiluted 1:200 in 40% FBS and a N. meningitidis medium (4×10⁹ cells/ml inPBS-Tween-20) was employed. The cell medium (0.2 ml) was filteredthrough a 0.2 μm pore polycarbonate membrane, followed by contacting themembrane for 10 min with 50 μl of the conjugate, followed by washingonce with PBS and once with water. The membrane was then introduced intothe measuring compartment. At 800 cells/ml, the observed voltage change(-210 μV/s) was 2.2× negative (-94), and at 8000 cells/ml (-936), 10×negative. Thus, ferrocyanide can be used in a sensitive method fordetection of organisms through a redox couple.

In the next study, a Genetran membrane was employed and the analyte wasa nucleic acid sample. Genetran is a positively-charged nylon membrane.The membrane was washed using 5% Triton in PBS, pH 7.0, 4×200 μl. Heatdenatured single-stranded calf thymus DNA in deionized water (50 μl) wasadded to the membrane. SSB-HRP conjugate solution (single-stranded DNAbinding protein was coupled using maleimide crosslinking chemistry tothiolated HRP) was diluted 1:15,000 in 0.2% BSA, PBS, 0.05% Tween-20,and 300 μl of the solution perfused through the membrane using aperistaltic pump, the filtration requiring 3-4 min. The membrane wasthen washed 3×200 μl with 5% Triton X-100 in PBS, followed by 3× 200 μlin peroxidase buffer. The membrane was then introduced into acompartment which contained the substrate solution comprising 25 μM ofRu⁺² chelate (same as previous example), 200 μM TMB, and 250 μM hydrogenperoxide in the previously described buffer. In the absence of DNA, thevoltage change was +262 μV/s, while at 150 pg, the voltage change was-63, at 600 pg, -2474, and at 2500 pg, - 16000. In a repeat of thedetermination, the negative control gave a voltage change of 150 μV/s,at +150 pg DNA, -312, at 600 pg DNA, -3116, and at 2500 pg DNA, -19268.

EXAMPLE 2

Detection of Alkaline Phosphatase

The apparatus employed was a redox sensor, which used a gold spot onsilicon water for the metal electrode as described in Example 1. Thefollowing reagents were employed:

1) Calf intestinal Alkaline Phosphatase

2) BCIP (5-Bromo-4-chloro-3-indolylphosphate, toluidine salt)

3) Alkaline phosphatase Buffer:

0.2 M Tris (Tris- hydroxymethyl!aminomethane)

1 mM MgCl₂, 0.05% NaN₃, 0.1% Tween 20, pH 10.0

The assay was based on using alkaline phosphotase as the enzyme, and thehydrolysis of indoxyl phosphate to indoxal. Indoxal is coupled to theinorganic redox component to generate a potentiometric signal.

The procedure employed was to add 10 μl of alkaline phosphatase solutionor buffer to 1 ml of substrate containing 1 mM indoxyl phosphate, 1 mMferricyanide and 0.5 mM ferrocyanide in 0.2 M Tris buffer, 1 mMmagnesium acetate, 0.05% sodium azide, pH 10.3. This solution wasquickly mixed and added to the measurement compartment. Measurementswere made every 0.18 min.

The buffer alone gave a +6.6 μV/s rate. The addition of BSA carrierprotein (2%) further increased the background rate to +16 μV/s.Increasing amounts of enzyme were added to the measuring compartment,starting at 7.8 ng/ml as the-concentration in the cell and increasing to124 ng/ml. The reaction rate, change in potential (μV/s) voltage, waslinear in the first 2 minutes of measurement. The reaction rateaccelerated as the ferricyanide was consumed, which occurred at the twohighest enzyme levels tested, 124 and 250 ng/ml. The reaction rate wasproportional to enzyme concentration from 7.8 to 250 ng/ml, a linearregression analysis gave a R² value of 0.997. An estimate of thesensitivity of the method was conservatively placed at 10 ng/ml.Reduction of the volume of the reaction cell to 0.1 μl should increasethe sensitivity to 1 pg of alkaline phosphatase. This represents8.3×10⁻¹⁸ moles of alkaline phosphatase or approximately 5×10⁶molecules.

It is evident from the above results, that by using an organic moleculecapable of being a substrate for an enzyme and capable of interactingwith a metallic redox pair, which in turn can affect the potential of ametal electrode on a semiconductor surface, great sensitivity can beachieved. In particular, hydrogen peroxide and TMB or indoxyl phosphatemay be used in conjunction with ferri- ferrocyanide or rutheniumchelates to couple alkaline phosphatase and peroxidase to a metalelectrode. These enzymes may be used as labels or in conjunction withother enzymes to measure the presence of specific analytes. Thus, highlysensitive assays for determining a wide variety of analytes and enzymesare obtained which may be carried out rapidly, accurately, with highsensitivity and in very small volumes.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. In a method for determining the presence of anoxidase in an assay medium by employing a metal electrode for detectionof a redox reaction, the improvement which comprises:(a) atetramethylbenzidine and hydrogen peroxide; with (b) an iron orruthenium containing ionic redox couple in the assay medium.
 2. A methodaccording to claim 1, wherein said oxidase is horseradish peroxidase andsaid hydrogen peroxide is produced in said assay medium by the enzymaticreaction of said oxidase with a substrate for the oxidase in said assaymedium.
 3. A method according to claim 1, wherein said oxidase ishorseradish peroxidase and said hydrogen peroxide is produced in theassay medium by a reaction of cholesterol oxidase or glucose oxidasewith a substrate for the oxidase.
 4. A method according to claim 1,wherein said iron containing ionic redox couple is hexacyanoferrateredox couple.
 5. A method according to claim 1, wherein said rutheniumcontaining ionic redox couple is ruthenium pentamine complex.
 6. In amethod for determining the presence of peroxidase in an assay medium byemploying a metal electrode for detection of a redox reaction, theimprovement which comprises:(a) a benzidine and hydrogen peroxide; with(b) an ionic redox couple containing an element selected from the groupconsisting of iron and ruthenium in the assay medium.
 7. A methodaccording to claim 6, wherein the metal is a noble metal.
 8. A methodaccording to claim 6, wherein the hydrogen peroxide is produced in theassay medium by an enzymatic reaction in the assay medium.
 9. In amethod for determining the presence of an oxidase in an assay medium byemploying a metal electrode for detection of a redox reaction, theimprovement which comprises:(a) a tetramethylbenzidine and hydrogenperoxide, with (b) an ionic redox couple containing an element selectedfrom the group consisting of iron and ruthenium in the assay medium. 10.A method according to claim 9, wherein the oxidase is horseradishperoxidase and the hydrogen peroxide is produced in the assay medium byan enzymatic reaction in the assay medium during the assay.
 11. A methodaccording to claim 9, wherein the oxidase is horseradish peroxidase andthe hydrogen peroxide is produced in the assay medium by a reaction ofcholesterol oxidase or glucose oxidase with a substrate for the oxidase.12. A method according to claim 9, wherein the ionic redox couplecontaining iron is a hexacyanoferrate redox couple.
 13. A methodaccording to claim 9, wherein the ionic redox couple containingruthenium is a ruthenium pentamine pyridine redox couple.
 14. In amethod for determining the presence of a hydrolase in an assay medium byemploying a metal electrode for detection of a redox reaction, theimprovement which comprises:(a) a compound selected from the groupconsisting of indoxyl ester and other; with (b) an ionic redox couplecontaining an element selected from the group consisting of iron andruthenium in the assay medium.
 15. A method according to claim 14,wherein the ionic redox couple containing ruthenium is rutheniumpentamine pyridine redox couple.
 16. A method according to claim 14,wherein the ionic redox couple containing iron is a hexacyanoferrateredox couple.
 17. A method according to claim 14, wherein the hydrolaseis alkaline phosphatase and the indoxyl ester is indoxyl phosphate.